Consolidated Bioprocessing: Synthetic Biology Routes to Fuels and Fine Chemicals

microorganisms Review Consolidated Bioprocessing: Synthetic Biology Routes to Fuels and Fine Chemicals Alec Banner, Helen S. Toogood * and Nigel S. Scrutton * E PSRC/BBSRC Future Biomanufacturing Research Hub, BBSRC/EPSRC Synthetic Biology Research Centre, SYNBIOCHEM Manchester Institute of Biotechnology, Department of Chemistry, School of Natural Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK; [email protected] * Correspondence: [email protected] (H.S.T.); [email protected] (N.S.S.); Tel.: +44-(0)161-3065152 (N.S.S.) Abstract: The long road from emerging biotechnologies to commercial “green” biosynthetic routes for chemical production relies in part on efficient microbial use of sustainable and renewable waste biomass feedstocks. One solution is to apply the consolidated bioprocessing approach, whereby microorganisms convert lignocellulose waste into advanced fuels and other chemicals. As lignocellulose is a highly complex network of polymers, enzymatic degradation or “saccharification” requires a range of cellulolytic enzymes acting synergistically to release the abundant sugars contained within. Complications arise from the need for extracellular localisation of cellulolytic enzymes, whether they be free or cell-associated. This review highlights the current progress in the consolidated bioprocessing approach, whereby microbial chassis are engineered to grow on lignocellulose as sole carbon sources whilst generating commercially useful chemicals. Future perspectives in the emerging biofoundry approach with bacterial hosts are discussed, where solutions to existing bottlenecks could potentially be overcome though the application of high throughput and iterative Design-Build-Test- Citation: Banner, A.; Toogood, H.S.; Scrutton, N.S. Consolidated Learn methodologies. These rapid automated pathway building infrastructures could be adapted for Bioprocessing: Synthetic Biology addressing the challenges of increasing cellulolytic capabilities of microorganisms to commercially Routes to Fuels and Fine Chemicals. viable levels. Microorganisms 2021, 9, 1079. https:// doi.org/10.3390/microorganisms9051079 Keywords: lignocellulose degradation; cellulases; biofoundry; consolidated bioprocessing; synthetic biology Academic Editors: Dietmar Haltrich and Daniel Kracher Received: 7 April 2021 1. Introduction Accepted: 14 May 2021 Many of the social and technological advances in the last century, from transportation Published: 18 May 2021 fuels to materials and pharmaceuticals, have been due to an increase in our understanding and utilisation of organic chemistry [1]. Much of this chemistry relies on the use of fossil Publisher’s Note: MDPI stays neutral carbon as synthons and is therefore inextricably coupled to the petrochemical industries. with regard to jurisdictional claims in published maps and institutional affil- These reactions often require high temperatures, high pressures and rare metal catalysts [1], iations. thereby generating polluting waste. Recognition of a global environmental crisis is in part driven by our over use and reliance on petroleum-based fuels and chemistries [2]. Alternative “green” synthetic routes have been developed, utilising non-fossil fuel-derived renewable biomass as synthons [3–13]. These emerging biotechnologies rely on the microbial conversion of biological carbon biomass (e.g., sugar cane; biomass waste streams) into Copyright: © 2021 by the authors. advanced synthetic fuels and bio-based chemistries [14]. A report into the development Licensee MDPI, Basel, Switzerland. of the bio-economy through to 2030 suggests biotechnological routes have the potential This article is an open access article to produce 75% of pharmaceutical or 35% of total chemicals currently made via synthetic distributed under the terms and conditions of the Creative Commons chemistry [15]. Attribution (CC BY) license (https:// Traditional genetic engineering routes to biocatalytic processes are increasingly being creativecommons.org/licenses/by/ superseded by synthetic biology technology, which employs a fermentative recombinant 4.0/). microbial approach to fine chemical production [5,16–20]. In this case, individual “parts” Microorganisms 2021, 9, 1079. https://doi.org/10.3390/microorganisms9051079 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 1079 2 of 20 of the introduced enzyme pathway(s) (e.g., enzyme homologues, promoters and ribosomal binding sites) are optimised to increase the flow through the pathway [21–23]. This process is often assisted by computer-aided-design programs to predict the optimal arrangement and sequence of each component [24]. This revolutionary approach allows for the development of de novo pathways to chemicals not found in nature, and can take advantage of enzyme engineering technologies to generate enzymes that catalyse novel reactions [25]. Examples of (bio)compounds produced by engineered microorganisms using a synthetic biology approach include artemisinic acid [26], β-farnesene [27], linalool [17,28,29], noscapine [30], butanol [31], 6-aminocaproic acid [32] and styrene [33]. The most complex to date was the complete synthesis of noscapine in Saccharomyces cerevisiae; an antitumor alkaloid derived naturally from Papever somniferum (opium poppy) [30]. In this case, eighteen heterologous enzymes were expressed in S. cerevisiae, of which only thirteen sequences were obtained from the native poppy. While the uptake of bio-based synthetic routes is increasing, significant advances are needed to increase the cost-effectiveness of these processes, to enable them to compete commercially with existing synthetic chemical or native biological routes [34]. As a result, few biosynthetic routes have reached industrial commercialisation, largely due to low product yields and the high cost of feedstocks. The largest scale commercial bioprod- uct is bioethanol produced from S. cerevisiae [35], with 29,000 million gallons generated worldwide in 2019 [36]. Most bioethanol is produced through anaerobic fermentation of glucose derived from either corn or sugarcane [37,38]. However, both crops are in direct competition with land use for food production. In a world where deforestation and famine are major issues, this has led some people to declare these fuels of little benefit compared to traditional fossil fuels [39]. A more environmentally sustainable solution is the utilisation of waste plant biomass or lignocellulose waste. Each year, around 200 billion tonnes of lignocellulosic waste are produced by industries such as farming and agriculture [40], and have limited commercial value. Typically, this waste would either be combusted, composted or used as a bulking agent in animal feed. The utilisation of this waste in synthetic biology applications could add commercial value to the waste and provide a carbon neutral source of fuels and other high value compounds. However, existing commercial microbial fermentations utilising lignocellulose waste as a carbon source rely on the release of the abundant recalcitrant sugars (e.g., glucose) via expensive pre-treatment strategies [41]. An alternative approach could be to employ a consolidated bioprocessing (CBP) strat- egy, whereby biocatalytic enzyme production, lignocellulose degradation (saccharification) and fermentation are accomplished within a single microorganism. This approach would likely reduce feedstock pre-processing requirements (and associated costs), making a more industrially viable and “green” process. To achieve this, either existing commercial strains require engineering to incorporate an extracellular localising cellulolytic system, or sec- ondary product biocatalytic pathways need to be integrated into naturally cellulolytic microorganisms. This review discusses current approaches and challenges for the utilisation of waste lignocellulose biomass as a feedstock for building a robust microbial chassis which can produce high value biomaterials, thereby enabling “green” solutions to biochemical production, and be competitive with chemical synthesis. We later propose the future application of tools developed by the rapidly expanding application of biofoundries, which have had a significant impact on the production of biosynthetic pathways, to the challenges of producing novel, cellulolytic biofactories. 2. Lignocellulose as a Carbon Source 2.1. Lignocellulose: A Heterogeneous Source of Polymeric Sugars Lignocellulose is potentially an ideal target as a low-cost carbon and energy source for microorganisms as it is the most abundant biologically derived polymer found in nature [14]. It is composed of an intricate species-specific network of cellulose (40–50%), Microorganisms 2021, 9, x FOR PEER REVIEW 3 of 20 2. Lignocellulose as a Carbon Source 2.1. Lignocellulose: A Heterogeneous Source of Polymeric Sugars Microorganisms 2021, 9, 1079 Lignocellulose is potentially an ideal target as a low-cost carbon and energy source3 of 20 for microorganisms as it is the most abundant biologically derived polymer found in nature [14]. It is composed of an intricate species-specific network of cellulose (40–50%), hemicellulose (20–40%) and lignin (20–35%). The hemicellulose interweaves with cellu- hemicelluloselose polymers, (20–40%) while the and lignin lignin content (20–35%). protec Thets the hemicellulose cellulose from interweaves degradation with [42]. cellulose The polymers,compact and while intertwining the lignin nature content of the protects

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